Research Summary G protein-coupled receptors (GPCRs) are an important superfamily of seven transmembrane proteins involved in cell-to-cell communication essential for sensing, movement, and thought processes. A significant portion of current approved drug therapies target GPCRs, but suffer from ?on-target? side-effects related to the engagement of differential signaling pathways known as ?functional selectivity? or ?biased signaling.? Exploiting biased signaling represents a promising approach toward designing pathway-selective drugs with better ?on- target? profiles, but the mechanisms at the structural level that lead to biased signaling are still poorly understood. Recent advancement in structural knowledge of biased ligand recognition has led to the identification of binding pocket motifs, such as extracellular loop 2 (EL2) and transmembrane (TM) 7, important for `switching' biased signaling via direct ligand engagement. Using a structure-based approach, my laboratory aims to use a variety of chemical biology and biophysical approaches to uncover common mechanisms within the binding pocket that govern biased signaling. Strategies will incorporate a combination of structure-guided mutagenesis, orthosteric/allosteric biased ligands, kinetic monitoring of G protein function and ?-arrestin recruitment using luciferase and bioluminescent resonance energy transfer (BRET) techniques, and structure- `functional selectivity' relationships (SFSRs) to ultimately to pin-point key GPCR binding motifs involved in effector switching. This approach focuses on key receptors where there is structural knowledge and G protein and ?-arrestin-biased ligands available. Through these studies, a comprehensive mechanistic understanding into GPCR biased signaling will guide researchers toward a new generation of superior therapeutics. 1